Alcohol (ethanol) is the most commonly used addictive substance in the United States with nearly 1 in every 12 adults suffering from alcohol abuse or dependence. Currently, ethanol is postulated to interact directly with alcohol-sensitive proteins thereby altering their function. The molecular mechanisms of target specificity and modulation are poorly understood. The G protein-gated inwardly rectifying potassium (GIRK) channels are directly activated by physiological concentrations (0.08% or ~18 mM) of ethanol, independent of their typical G- protein mediated pathway. Recent studies identified an alcohol pocket within the cytoplasmic domain of the potassium channel, which was shown to be critical for the GIRK channel activation by alcohol. However, there is accumulating evidence demonstrating the increasingly important role of the lipid environment on channel gating. The alcohol-mediated effects of the lipid environment on GIRK channels are of particular interest because the membrane associated molecule PIP2 is required for activation by both alcohol and G-proteins. Furthermore, alcohol has both acute and chronic effects on membrane content and distribution of cholesterol, which is ubiquitous in cell membranes and also modulates GIRK channel activity. In order to fully understand the effects of alcohol and develop effective treatments, it is essential to elucidate the molecular and structural mechanisms by which alcohol modulates GIRK channels in their lipid environment. It is hypothesized that alcohol and cholesterol both potentiate GIRK channel activity independently through direct channel interactions. Most studies to date have studied channels expressed in heterologous expression systems, where the lipid composition is pre-determined and difficult to manipulate. To overcome this obstacle in the field, GIRK channels will be purified and reconstituted in a system that allows for manipulation of lipid composition and constituents. A fluorescence-based flux assay for measuring macroscopic channel activity and single channel recordings of channels in lipid bilayers will be used to (i) determine the alcohol sensitivity of purified GIRK2 channels in proteoliposomes, (ii) test the hypothesis that cholesterol and alcohol modulate the GIRK2 channels by independent mechanisms, and (iii) elucidate the mechanism by which alcohol and cholesterol affect GIRK2 channel gating. These experiments will provide critical details on the mechanism underlying alcohol activation of GIRK channels and the role that the lipid environment plays in this modulation. Understanding the mechanism of this process is paramount to developing selective pharmacological tools that could be used in the treatment of alcohol abuse and addiction.